1-methyl-6-oxyindole-2-formamide derivative as well as preparation method and application thereof

Abstract

The invention provides a 1-methyl-6-oxyindole-2-formamide derivative as well as a preparation method and application thereof, and belongs to the field of drug synthesis, the structural formula of the 1-methyl-6-oxyindole-2-formamide derivative is shown as a general formula I: # imgabs0, R1 is selected from # imgabs1, # imgabs2, # imgabs3, G = C or N, R2 is selected from a group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 8, 9, 10, 12, 13, 14, 16, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17 W = C or N. The 1-methyl-6-oxoindole-2-formamide mother nucleus group with biological activity contained in the compound is further chemically modified to generate a plurality of compounds with higher biological activity, so that the wide application of the compounds in biological medicines and the development prospect of pharmaceutical preparations are expanded. The compound can significantly inhibit proliferation of breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma and other cells at a low dosage (nanomole), and can effectively inhibit STAT3 protein phosphorylation, which shows that the compound has a prospect of being developed into antitumor drugs.

Description

Technical Field

[0001] The invention belongs to the field of drug synthesis, and specifically relates to a 1-methyl-6-oxyindole-2-carboxamide derivative, a preparation method and application thereof. Background Art

[0002] Cancer has become a major cause of premature death and shortened life span in the global population. Although there have been great advances in the treatment of cancer clinically, due to the heterogeneity and continuous evolution of tumors, the response rate of most drugs is low and they are prone to drug resistance, resulting in a severe lack of effective therapeutic drugs. Conventional cytotoxic chemotherapy drugs such as paclitaxel, cisplatin, carboplatin and capecitabine have the advantages of a broad anti-cancer spectrum and good efficacy, but they have large toxic side effects and are prone to early drug resistance. Therefore, actively exploring and developing new anti-cancer drugs is of great clinical significance.

[0003] The present application synthesized a class of 1-methyl-6-oxyindole-2-carboxamide derivatives with a new structural formula. Through some biological technical analysis, it was found that this class of compounds can significantly inhibit the proliferation of breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma and other cells at extremely low doses, and can effectively inhibit STAT3 protein phosphorylation. Therefore, further development of this class of compounds will be of great significance in the application of tumor treatment. Summary of the invention

[0004] The purpose of the present invention is to provide a 1-methyl-6-oxyindole-2-carboxamide derivative, a preparation method and application thereof.

[0005] Based on the above purpose, the present invention adopts the following technical solutions:

[0006] A 1-methyl-6-oxyindole-2-carboxamide derivative, the structural formula of which is shown in general formula I:

[0007]

[0008] Wherein R1 is selected from

[0009] G = C or N;

[0010] W=C or N.

[0011] That is, the above-mentioned 1-methyl-6-oxyindole-2-carboxamide derivative is specifically a compound with the following structure:

[0012]

[0013] A biologically acceptable salt formed by the above-mentioned 1-methyl-6-oxyindole-2-carboxamide derivative and at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, ascorbic acid, boric acid, lactic acid and ethylenediaminetetraacetic acid.

[0014] The preparation method of the above-mentioned 1-methyl-6-oxyindole-2-carboxamide derivative, the synthetic route is as follows:

[0015]

[0016] The specific synthesis steps are as follows:

[0017] (1) Compound 1, Compound 2, HBTU and DIEA were dissolved in DMF. After the reaction was completed by stirring at room temperature, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was spin-dried and slurried with ethyl acetate, filtered, and the solid was dried to obtain Compound 3;

[0018] (2) Compound 3, Compound 4 and potassium carbonate were dissolved in DMF, stirred at 70-90° C. and after the reaction was complete, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was spin-dried and slurried with ethyl acetate, filtered, and the solid was dried to obtain Compound 5;

[0019] (3) Compound 5, compound 6, Pd(PPh3)2Cl2, CuI and triethylamine were dissolved in DMF, stirred at 70-90°C until the reaction was complete, and then the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was spin-dried and slurried with ethyl acetate, filtered, and the solid was dried to obtain the compound of formula I.

[0020] Furthermore, in step (1), the molar ratio of compound 1, compound 2, HBTU and DIEA is 1:1:(1-1.5):3; in step (2), the molar ratio of compound 3, compound 4 and potassium carbonate is 1:(1-1.5):(1-1.5); in step (3), the molar ratio of compound 5, compound 6, Pd(PPh3)2Cl2, CuI and triethylamine is 1:(1-1.5):(0.01-0.03):(0.01-0.09):5.

[0021] Use of the above-mentioned 1-methyl-6-oxyindole-2-carboxamide compounds and biologically acceptable salts thereof in the preparation of STAT3 protein inhibitors.

[0022] Application of the above-mentioned 1-methyl-6-oxyindole-2-carboxamide derivatives and biologically acceptable salts thereof in the preparation of anti-tumor drugs.

[0023] Preferably, the anti-tumor drug refers to a drug for treating breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma, etc.

[0024] Specifically, the present invention synthesizes a class of 1-methyl-6-oxindole-2-carboxamide derivatives with a new structure, RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, RDG-1018, etc. The proliferation inhibition effect of such compounds on various cancer cells was detected by CCK-8 method; and the phosphorylation of STAT3 protein can be effectively inhibited.

[0025] The results show that the compounds RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, and RDG-1018 of the present invention can effectively inhibit the proliferation of breast cancer, liver cancer, pancreatic cancer, gastric cancer and other cells, and can effectively inhibit the phosphorylation of STAT3 protein.

[0026] In summary, the present invention provides a new 1-methyl-6-oxyindole-2-carboxamide derivative and the use and potential molecular mechanism of its derivatives in tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the result of the effect of different concentrations of RDG-1001 on the protein expression of MDA-MB-468 cells. DETAILED DESCRIPTION

[0028] In order to make the technical purpose, technical solution and beneficial effects of the present invention more clear, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0029] In the method for synthesizing the compound of formula I of the present invention, various raw materials used in the reaction can be prepared by those skilled in the art according to prior knowledge, or can be prepared by methods known in the literature, or can be purchased commercially. The intermediates, raw materials, reagents, reaction conditions, etc. used in the above reaction schemes can be appropriately changed according to the prior knowledge of those skilled in the art.

[0030] In the present invention, unless otherwise specified, (i) the temperature is expressed in degrees Celsius (°C), and the operation is carried out at room temperature; more specifically, the room temperature refers to 20-30°C; (ii) the organic solvent is dried by a common drying method, and the solvent is evaporated by a rotary evaporator under reduced pressure, and the bath temperature is not higher than 50°C; the developing agent and the eluent are both in volume ratio; (iii) the reaction process is tracked by thin layer chromatography (TLC); (iv) the final product has a satisfactory proton nuclear magnetic resonance ( 1 H-NMR).

[0031] Example 1: The synthesis of all compounds is as follows:

[0032] The specific synthesis method takes compound RDG-1001 as an example, and the structural formula is as follows:

[0033]

[0034] The name of compound RDG-1001 is (1-methyl-6-((5-(3-morpholinoprop-1-yn-1-yl)pyrazin-2-yl)oxy)-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone,

[0035] Its synthetic route is as follows:

[0036]

[0037] Step 1. (6-hydroxy-1-methyl-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone (Compound 3)

[0038] Compound 1 (2.0 g, 10.63 mmol, 1.0 eq), compound 2 (2.92 g, 10.63 mmol, 1.0 eq), HBTU (4.85 g, 12.57 mmol, 1.2 eq) and DIEA (4.15 g, 31.38 mmol, 3.0 eq) were dissolved in 30 mL DMF, stirred at room temperature for 3 hours, and the reaction was completed by TLC monitoring. The reaction solution was diluted with 200 mL ethyl acetate, washed three times with saturated brine (200 mL*3), the organic phase was dried and spin-dried, and purified by slurrying with 15 mL ethyl acetate and filtered, and the solid was collected and dried in an oven at 80°C to obtain 4.20 g of white solid compound 3, with a yield of 88.9%.

[0039] 1 H NMR (CDCl3, 300MHz) δ: 9.02 (s, 1H), 8.25 (d, J = 8Hz, 1H), 7.78 (d, J = 8Hz, 1H), 7.53 (m, 2H), 7.22 (d, J = 8Hz, 1H), 7.18 (d ,J=8Hz,2H),6.86(d,J=8Hz,1H),4.32(m,2H),4.20-4.13(m,3H),3.95-3.88(m,2H),2.85(s,3H),3.04-2.96(m,5H).

[0040] Step 2. (6-((5-bromopyrazin-2-yl)oxy)-1-methyl-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone (Compound 5)

[0041] Compound 3 (5.0 g, 11.17 mmol, 1.0 eq), compound 4 (2.69 g, 11.17 mmol, 1.0 eq) and potassium carbonate (1.85 g, 13.41 mmol, 1.2 eq) were dissolved in 50 mL DMF, stirred at 80 ° C for 2 hours, and the reaction was completed by TLC monitoring. The reaction solution was diluted with 300 mL ethyl acetate, washed three times with saturated brine (200 mL * 3), the organic phase was dried and slurried with 18 mL ethyl acetate, filtered and the filter cake was collected, and the filter cake was dried to obtain 5.20 g of yellow solid compound 5, with a yield of 71.4%.

[0042] 1H NMR(DMSO-d6,300MHz)δ:8.79(d,J=1.2Hz,1H),8.67(d,J=1.2Hz,1H),7.69(d,J=8.6Hz,1H),7.51(d,J=1.9Hz,1H),7.32(s,1H),7.30(s,1H), 7.05(s,1H),7.04(s,1H),7.02(dd,J=8.5,2.1Hz,1H),7.01(s,1H),4.7 6(d,J=8.9Hz,2H),3.74(s,3H),3.67(s,4H),3.51(s,2H),2.44(s,4H).

[0043] Step 3.(1-methyl-6-((5-(3-morpholinoprop-1-yn-1-yl)pyrazin-2-yl)oxy)-1H-indol-2-yl)(4-(4-

[0044] (2,2,2-

[0045] trifluoroethoxy)benzyl)piperazin-1-yl)methanone(RDG-1001)

[0046] Compound 5 (200 mg, 0.33 mmol, 1.0 eq), compound 6 (45.56 mg, 0.36 mmol, 1.1 eq), Pd(PPh3)2Cl2 (4.56 mg, 0.006 mmol, 0.02 eq), CuI (3.15 mg, 0.016 mmol, 0.05 eq) and TEA (167 mg, 1.65 mmol, 5.0 eq) were dissolved in 10 mL DMF, stirred at 80 ° C for 48 hours, and the reaction was completed by TLC monitoring. The reaction solution was diluted with 300 mL ethyl acetate, washed three times with saturated brine (200 mL * 3), the organic phase was dried and spin-dried, and the crude product was purified by column (volume ratio, dichloromethane / methanol = 70 / 1 to 20 / 1) to obtain 182 mg of yellow solid compound RDG-1001, with a yield of 85.0%.

[0047] 1H NMR(CDCl3,400MHz)δ:8.37(d,J=8.0Hz,1H),8.18(d,J=8.0Hz,1H),7.63(d,J=8.0Hz,1H),7.28(d,J=8.0Hz,3H),7.16(s,1H),6.93-6.91(m ,2H),6.60(s,1H),4.38-4.32(q,J=16Hz,J=16Hz,2H),3.80-3.75(m,8H),3.56-3.49(m,4H),2.65(t,J=4Hz,3H),2.49(m,3H),1.60(m,5H).

[0048] The synthesis of RDG-1001, RDG-1002, RDG-1003, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, and RDG-1018 can refer to the method of Example 1, except that in the last step, the morpholine group of compound 6 is replaced with an alkynyl compound of the corresponding substituent.

[0049] The synthesis of RDG-1004 and RDG-1005 can refer to the method of Example 1, except that in the second step, compound 4 is replaced with the dibromo group of the corresponding heterocycle, and in the last step, the morpholine group of compound 6 is replaced with the alkynyl compound of the corresponding substituent.

[0050] Example 2: Proliferation inhibition of RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, RDG-1018 on breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma and other cells

[0051] MDA-MB-468, HepG2, BxPC-3, SGC7901, H460, KYSE450, Hela, MM.1S, and OCI-LY3 cells in the logarithmic growth phase were collected and counted, and the cell suspension concentration was adjusted to 5×10 4 / mL, added to a 96-well cell culture plate, with a volume of 100 μL per well. Using DMSO as a solvent control, the compounds RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, and RDG-1018 described in the present invention were diluted with DMSO and added to the culture wells, so that the final concentrations of the compounds in the system were 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 (μmol / L), respectively. After 72 hours of continuous culture, 10 / 20 μL of CCK-8 solvent was added to each well, incubated at 37°C for 3 hours, and the OD value at an absorption wavelength of 450 nm was measured by an ELISA reader. The results were recorded, and the cell growth curve was drawn with the dose of the compound as the abscissa and the absorbance value as the ordinate. The statistical results of the half inhibition rate (IC50 value) of the compound on tumor cells are shown in Table 1 below:

[0052] Table 1. CCK-8 assay of the inhibitory effect of RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, RDG-1018 on the proliferation of breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma and other cells

[0053]

[0054]

[0055]

[0056] The table shows that RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, and RDG-1018 have good proliferation inhibitory effects on cells such as breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, and diffuse large B-cell lymphoma, especially in breast cancer, multiple myeloma, and diffuse large B-cell lymphoma cells. The tumor inhibitory activity is stronger. This application uses RDG-1001 as an example to conduct a preliminary study on the anti-tumor mechanism of this type of compound.

[0057] Example 3: Inhibitory effect of RDG-1001 on STAT3 phosphorylation and CyclinD1 expression in MDA-MB-468 cells

[0058] 1. Cell culture and drug addition: a. Take MDA-MB-468 cells in the logarithmic growth phase and adjust the density to 2×10 5 indivual

[0059] / mL single cell suspension, 2mL cell suspension per well was inoculated into 6-well plates. b. Incubate in a 37℃ incubator overnight, add different concentrations of RDG-1001 (final concentration is 0, 10, 30, 60, 100nM), DMSO as negative control, 30nM RDf001 [Chinese full name: 1-methyl-6-((5-((4-(trifluoromethyl)phenyl)ethynyl)pyrazin-2-yl)oxy)-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone] as positive control. c. After culturing for 24h, cells were lysed with RIPA lysis buffer and proteins were collected.

[0060] 2. Cell collection and lysis: a. Discard the upper culture medium and wash the cells twice with pre-cooled PBS. Add 100μL of pre-cooled RIPA cell lysis buffer (protease inhibitors and PMSF are added to the lysis buffer in a ratio of 1:100 in advance and mixed) to each well. b. Lyse on ice for 3 minutes, scrape the cells with a cell scraper and collect them in a 1.5mL EP tube; place on ice for lysis for 30 minutes, and vortex every 6 minutes. c. Centrifuge at 4℃, 12000g for 10 minutes. d. Transfer the cell supernatant to a new EP tube. d. The cell supernatant is divided into two parts: take 5μL and add it to a 1.5mL EP tube for BCA protein content measurement, then add 45μL of 1×PBS and mix well for use; take 80μL of the remaining cell supernatant, add 20μL of 5×SDS Loading Buffer, mix well, boil in boiling water for 10 minutes, centrifuge and load or store in a -20℃ refrigerator.

[0061] e. Protein concentration determination steps: (1) Preparation of BCA working solution: Calculate the total amount of A and B mixed working solution required based on the number of standards and samples to be tested. Prepare the working solution at a volume ratio of 50:1 between BCA reagent A and B, vortex and mix well for later use.

[0062] (2) 1× PBS diluted protein standard:

[0063]

[0064]

[0065] (3) Take 25 μL of each of the protein standard solution and the sample supernatant diluted with PBS (10-fold dilution) and add them to a new 96-well plate. Then add 200 μL of the pre-prepared BCA working solution and mix thoroughly. Be careful not to blow and create bubbles. Cover the 96-well plate tightly and react in a 37°C incubator for 30 minutes.

[0066] (4) Take out the 96-well plate and restore it to room temperature for 3-5 minutes. Measure the absorbance value of A562 on the microplate reader and copy the obtained value and save it in an Excel table. Make a standard curve and calculate the protein content of 1 μL of each sample for protein loading. 3. SDS-PAGE: (1) Fix the gel plate and prepare 10% SDS-PAGE separation gel.

[0067] Prepare separation gel according to the following table: 10 mL

[0068] Deionized water 4.0mL 30%(m / v)Acrylamide 3.3mL 1.5M Tris-HCl (PH 8.8) buffer 2.5mL 10%(m / v)SDS 0.1mL 10%(m / v)APS 0.1mL TEMED 4μL Total 10mL

[0069] (2) Add the mixed separation gel to two gel plates, add to a position 1.0 cm from the top, fill the gel plates with anhydrous ethanol, and let stand for 30 to 45 minutes.

[0070] (3) After separation and gelation, pour out the remaining anhydrous ethanol and absorb it with filter paper.

[0071] (4) Prepare 5 mL of 5% concentrated gel according to the following table

[0072] Deionized water 2.77mL 30%(m / v)Acrylamide 830μL 0.5M Tris-HCl (PH 6.8) buffer 1.26mL 10%(m / v)SDS 50μL 10%(m / v)APS 50μL TEMED 5μL Total 5mL

[0073] (5) Slowly add the prepared concentrated glue to the glue plate to avoid generating bubbles, insert a comb, and let it stand for 30 to 45 minutes.

[0074] (6) Take out the protein sample, heat it in a water bath at 100°C for 5 min, and centrifuge it at 10,000 rpm for 5 min.

[0075] (7) Fix the gel plate into the electrophoresis tank, add SDS-PAGE electrophoresis buffer, remove the comb, and add the processed protein samples into the sample tank in sequence, with 50 μg of protein per well.

[0076] (8) Electrophoresis at 80 V for 40 min.

[0077] (9) Change the voltage to 120 V and run the electrophoresis for about 1.5 h until the bromophenol blue runs out of the colloid.

[0078] IV. Western-blot: (1) Rinse the SDS-PAGE gel after electrophoresis in TBST buffer once, and soak the protein gel in transfer buffer. (2) Soak a layer of cotton pad in membrane transfer buffer, clamp it to the transfer instrument with tweezers, and place it in the order of blackboard, cotton pad, filter paper, protein gel, PVDF membrane, filter paper, cotton pad and white board, clamp it, and put it into the transfer instrument. If there are bubbles between each layer, use a glass tube to gently roll them out. (3) Turn on the transfer instrument and transfer at 300mA constant current for 80min. (4) Put the membrane in TBST buffer and rinse 3 times, 8min each time. (5) Block with 20mL of 5% BSA-TBST blocking solution at room temperature for 2h. (6) Add primary antibody and incubate overnight at 4℃60rpm. (7) Wash the membrane three times with TBST at room temperature, shaking at 60rpm, 10min each time. (8) Add secondary antibody and incubate at room temperature for 1h. (9) Wash the membrane three times with TBST at room temperature, shaking at 60 rpm, for 10 min each time. (10) Take 1 mL each of chemiluminescent substrate solution A and solution B, and color develop at room temperature for 2 min. (11) Use filter paper to absorb the liquid on the membrane and expose it to light.

[0079] 5. Reagent preparation:

[0080] (1) 10% SDS: Weigh 1 g of high-purity (electrophoresis grade) SDS into a 10 mL centrifuge tube, add about 8 mL of deionized water, heat to dissolve, dilute to 10 mL, and store at room temperature.

[0081] (2) 10% ammonium persulfate (AP): weigh 1 g of ammonium persulfate, add about 10 mL of deionized water, stir to dissolve, and store at 4°C.

[0082] (3) 5× electrophoresis buffer: weigh 15.1 g of Tris, 94 g of Glycine, and 5.0 g of SDS into a beaker, add 1 L of double distilled water to dissolve, store at room temperature, and dilute 5 times before use.

[0083] (4) Transfer buffer: Weigh 5.8 g of Tris, 11.6 g of glycine, and 0.75 g of SDS into a beaker, add 700 mL of double distilled water, dissolve and adjust the volume to 800 mL, and finally add 200 mL of methanol.

[0084] (5) 1.5 mol / L Tris-HCl, 100 mL: Dissolve 18.15 g Tris base in 80 mL water and adjust the pH to 8.8 with 4 N HCl. Make up to 100 mL.

[0085] (6) 0.5 mol / L Tris-HCl, 1000 mL: Weigh 60.5 g of Tris base, add water to 850 mL, add concentrated hydrochloric acid and stir until completely dissolved, then adjust the pH to 6.8 and add water to 1 L.

[0086] (7) TBS buffer: Weigh 8.8 g of NaCl and dissolve it in 800 mL of distilled water. Add 10 mL of 1 mol / L Tris-HCl (pH 7.5) to make up to 1 L and store at room temperature.

[0087] (8) TBST buffer: Add 500 μL of 20% Tween 20 to 1 L of TBS buffer to make the final concentration of Tween 20 0.1%. Prepare it before use.

[0088] (9) Blocking solution, antibody diluent: Add 5% skim milk powder or BSA to TBST buffer and prepare it before use.

[0089] The above test results are detailed in Figure 1 ,like Figure 1 The results showed that treatment with 30nM, 60nM, and 100nM of RDG-1001 could effectively downregulate the expression levels of p-STAT3(Y705), p-STAT3(S727), and STAT3's downstream target protein CyclinD1.

[0090] The above results show that RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, RDG-1018 can significantly inhibit the proliferation of breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma and other cells, and can effectively downregulate the expression levels of p-STAT3 (Y705), p-STAT3 (S727) and STAT3's downstream target protein CyclinD1. Therefore, this type of drug has good anti-cancer effect and development potential.

[0091] According to the general approach of drug development (conventional anti-tumor in vitro screening first, then targeted research), the compounds of the present invention can be applied to cancer treatment drugs related to abnormal cell proliferation, and can be prepared as anti-tumor drugs by mixing with human-acceptable salts or with pharmaceutical carriers.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate but not limit the technical solutions of the present invention, and any equivalent substitutions of the present invention and any modifications or partial substitutions that do not depart from the spirit and scope of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A 1-methyl-6-oxyindole-2-carboxamide derivative, characterized in that: The structural formula is shown in general formula I: G = C or N; W=C or N.

2. A biologically acceptable salt of the 1-methyl-6-oxyindole-2-carboxamide derivative of claim 1 and at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, ascorbic acid, boric acid, lactic acid and ethylenediaminetetraacetic acid.

3. The method for preparing the 1-methyl-6-oxyindole-2-carboxamide derivative according to claim 1, characterized in that: The synthetic route is as follows: The specific synthesis steps are as follows: (1) Compound 1, Compound 2, HBTU and DIEA were dissolved in DMF. After the reaction was completed by stirring at room temperature, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was spin-dried and slurried with ethyl acetate, filtered, and the solid was dried to obtain Compound 3; (2) Compound 3, Compound 4 and potassium carbonate were dissolved in DMF, stirred at 70-90° C. and after the reaction was complete, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was spin-dried and slurried with ethyl acetate, filtered, and the solid was dried to obtain Compound 5; (3) Compound 5, compound 6, Pd(PPh3)2Cl2, CuI and triethylamine were dissolved in DMF, stirred at 70-90°C until the reaction was complete, and then the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was spin-dried and slurried with ethyl acetate, filtered, and the solid was dried to obtain the compound of formula I.

4. The method for preparing the 1-methyl-6-oxyindole-2-carboxamide derivative according to claim 3, characterized in that: In step (1), the molar ratio of compound 1, compound 2, HBTU and DIEA is 1:1:(1-1.5):3; in step (2), the molar ratio of compound 3, compound 4 and potassium carbonate is 1:(1-1.5):(1-1.5); in step (3), the molar ratio of compound 5, compound 6, Pd(PPh3)2Cl2, CuI and triethylamine is 1:(1-1.5):(0.01-0.03):(0.01-0.09):

5.

5. Use of the 1-methyl-6-oxyindole-2-carboxamide derivative according to claim 1 or the biologically acceptable salt of the 1-methyl-6-oxyindole-2-carboxamide derivative according to claim 2 in the preparation of a STAT3 protein inhibitor.

6. Use of the 1-methyl-6-oxyindole-2-carboxamide derivative according to claim 1 or the biologically acceptable salt of the 1-methyl-6-oxyindole-2-carboxamide derivative according to claim 2 in the preparation of an anti-tumor drug, characterized in that: The anti-tumor drugs are drugs for treating breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma and diffuse large B-cell lymphoma.

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